Ma Haiyan, Bell Kristin N, Loker Rossi N
Northern Biomolecular Services, 4717 Campus Drive, Kalamazoo, MI 49008, USA.
Mol Ther Methods Clin Dev. 2020 Nov 17;20:152-168. doi: 10.1016/j.omtm.2020.11.007. eCollection 2021 Mar 12.
Gene and cell therapy fields have experienced remarkable growth during the past decade. Demands for preclinical and clinical safety assessments of these cell and gene therapy test articles (TAs) have effectively increased the necessity for regulated biodistribution, vector shedding, gene expression, and/or pharmacokinetics bioanalysis studies. Guidance documents issued from numerous international regulatory authorities recommend the use of quantitative polymerase chain reaction (qPCR) and/or quantitative reverse transcriptase PCR (qRT-PCR) assays due to their highly sensitive and robust target-specific detection. However, only preclinical biodistribution assay sensitivity is specified in these documents. Criteria such as accuracy, precision, and repeatability are not yet defined. This guidance void has resulted in several conflicting institutional interpretations of essential parameters necessary for the development and validation of robust assays to support safety assessments of gene and cell therapy TAs. There is an urgent need for an ongoing discussion among bioanalytical scientists in this field to generate a "best practice" consensus around preclinical and clinical qPCR/qRT-PCR assay design. With regard to this need, we offer critical points to consider when developing, validating, running sample analysis, and reporting qPCR/qRT-PCR assays.
在过去十年中，基因和细胞治疗领域经历了显著的发展。对这些细胞和基因治疗试验品（TAs）进行临床前和临床安全性评估的需求，有效地增加了对规范的生物分布、载体脱落、基因表达和/或药代动力学生物分析研究的必要性。众多国际监管机构发布的指导文件推荐使用定量聚合酶链反应（qPCR）和/或定量逆转录酶PCR（qRT-PCR）检测方法，因为它们具有高度灵敏且强大的靶向特异性检测能力。然而，这些文件中仅规定了临床前生物分布检测的灵敏度。诸如准确性、精密度和可重复性等标准尚未明确。这种指导空白导致了对用于支持基因和细胞治疗试验品安全性评估的稳健检测方法的开发和验证所需基本参数的几种相互矛盾的机构解释。该领域的生物分析科学家迫切需要进行持续讨论，以围绕临床前和临床qPCR/qRT-PCR检测设计达成“最佳实践”共识。关于这一需求，我们提供了在开发、验证、进行样本分析和报告qPCR/qRT-PCR检测时需要考虑的关键点。
